Rapid thermal cycling for sample analyses and processing

ABSTRACT

Apparatus and method for thermal processing of nucleic acid in a thermal profile is provided. The apparatus employs a reactor holder for holding reactor(s) each accommodating reaction material containing the nucleic acid. The apparatus comprises a first bath; and a second bath, bath mediums in the baths being respectively maintainable at two different temperatures T HIGH  and T LOW ; and a transfer means for allowing the reactor(s) to be in the two baths in a plurality of thermal cycles to alternately attain: a predetermined high target temperature T HT , and a predetermined low target temperature T LT , while the apparatus adapts to a temperature-offset feature defined by at least one condition from the group consisting: a) the T HT  is lower than the T HIGH , b) the T LT  is higher than the T LOW , and c) the conditions at a) and b).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the national phase entry of InternationalApplication No. PCT/SG2017/050293, filed on Jun. 9, 2017, which is basedupon and claims priority to U.S. Patent Application No. 62/348,155,filed on Jun. 10, 2016 and Singapore Patent Application No.10201700260X, filed on Jan. 12, 2017, the entire contents of which areincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a method and an apparatus forperforming amplification reaction of nucleic acids in a sample.

BACKGROUND

Polymerase chain reaction (PCR) is increasingly important to molecularbiology, food safety and environmental monitoring. A large number ofbiological researchers use PCR in their work on nucleic acid analyses,due to its high sensitivity and specificity. The time cycle of a PCR istypically in the order of an hour, primarily due to a time-consuming PCRthermal cycling process that is adapted to heat and cool reactorscontaining the sample to different temperatures for DNA denaturation,annealing and extension. Typically, the thermal cycling apparatus andmethod employs moving the reactors between two heating baths whosetemperatures are set at the target temperatures as required for nucleicacid amplification reactions. Researchers have been constantly strivingto increase the speed of thermal cycling.

Thermoelectric cooler (TEC) or Peltier cooler is also used as theheating/cooling element. However, it provides a typical ramping rate of1-5 degree C./sec which is rather slow in changing the temperature ofthe reactor and disadvantageously increases the time of the thermalcycling.

As an attempt to increase the PCR speed by reducing thermal mass,microfabricated PCR reactor with embedded thin film heater and sensorwas developed to achieve faster thermal cycling at a cooling rate of 74degree Celsius/s and a heating rate of around 60-90 degree Celsius/s.However, such a wafer fabrication process for making the PCR device isextremely expensive and thus is impractical in meeting the requirementof low cost disposable applications in biological testing.

Hot and cold air alternately flushing the reactors in a closed chamberto achieve higher temperature ramping than the TEC-based thermal cyclerhas been described. However, from the heat transfer point of view, airhas much lower thermal conductivity and heat capacity than liquid, hencethe temperature ramping of the air cycler is slower than that with aliquid. The TEC needs a significant amount of time to heat and coolitself and the heat block above the TEC. Further there is also need toovercome the contact thermal resistance between the heat block and thereactors.

Alternating water flushing cyclers were also developed in which water oftwo different temperatures alternately flush the reactors to achieve PCRspeed. However, such devices contain many pumps, valves and tubingconnectors which increase the complexity of maintenance and lower thereliability while dealing with high temperature and high pressure. Withcirculating liquid bath medium, the liquid commonly spills out from thebaths.

Traditional water bath PCR cyclers utilize the high thermal conductivityand heat capacity of water to achieve efficient temperature heating andcooling. But, such cyclers have large heating baths containing a largevolume of water which is hard to manage in loading and disposal, andalso makes the heating time to target temperatures too long beforethermal cycling can start. Such cyclers also have large device weightand high power consumption. The water tends to vaporize with usage andneeds to be topped up. Besides, during the thermal cycling every timethe reactor is alternately inserted into the baths, a layer of waterremains adhered on the reactor body when taken out of each bath, therebycausing the change in temperature inside the reactor to get slowerundesirably.

Researchers also tested moving heated rollers of different temperaturesto alternately contact the reactors. However, use of long tubingreactors make it not only cumbersome to install and operate a largearray of reactors, but also expensive. When the reactors are in a largearray or a panel, it may be challenging to achieve heating uniformityamong all the reactors.

The present invention provides an improved method and apparatus forenabling thermal cycling nucleic acid at an ultra-fast speed ataffordable cost without using complex and expensive components orconsumables. The apparatus is robust, light weight, easy to use, needs asmall amount of bath medium in the baths and can handle disposablereactors for the reaction material to avoid cross contamination from onereactor to the next. This invention provides a great positive impact onbiological analysis.

SUMMARY

Unless specified otherwise, the term “comprising” and “comprise” andgrammatical variants thereof, are intended to represent “open” or“inclusive” language such that they include recited elements but alsopermit inclusion of additional, unrecited elements. The terminologies‘first bath’, ‘second bath’ . . . ‘sixth bath’ do not constitute thecorresponding number of baths in a sequence but merely are names forease of identification with respect to the purpose they serve. Thesebaths may not represent separate physical entities as some of them maybe sharable. The term ‘thermal processing’ includes: a) thermal cycling,and optionally includes: b) thermal process steps before and/or afterthermal cycling. The term ‘thermal profile’ refers to thetemperature-time variation of the reactor(s) during a) alone or duringa) with b).

According to a first aspect, an apparatus is provided for thermalprocessing of nucleic acid in a thermal profile, the apparatus employinga reactor holder for holding reactor(s) each accommodating reactionmaterial containing the nucleic acid and the reactor(s) being in anyform such as tube(s) or wellplate(s) or chip(s) or cartridge(s), theapparatus comprising: a first bath; and a second bath, bath mediums inthe baths being respectively maintainable at two different temperaturesT_(HIGH) and T_(LOW); and a transfer means for allowing the reactor(s)to be in the two baths in a plurality of thermal cycles to alternatelyattain: a predetermined high target temperature T_(HT), and apredetermined low target temperature T_(LT), while the apparatus adaptsto a temperature-offset feature defined by at least one condition fromthe group consisting: a) the T_(HT) is lower than the T_(HIGH), b) theT_(LT) is higher than the T_(LOW), and c) the conditions at a) and b),the transfer means being operable by at least one mode from the groupconsisting: a temperature guided motion controlling means (TeGMCM) thatis operable based on the real-time temperature as sensed by a reactortemperature sensor during thermal cycling, and a time guided motioncontrolling means (TiGMCM) that is operable based on the time-periodsfor which the reactor(s) are allowed to be in the baths, the bath mediumin any of the baths being in any phase including air, liquid, solid,powder and a mixture of any of these. Advantageously herein the conceptof Newton's law is made use of which states that the rate of heat lossof a body is proportional to the difference in temperatures between thebody and its surroundings. By maintaining the first bath temperature atT_(HIGH) that is significantly higher than the T_(HT) and the secondbath temperature at T_(LOW) that is significantly lower than the T_(LT),the reactor(s) can undergo thermal cycling significantly faster. TheTiGMCM can be user calibrated for the time-periods. TeGMCM allows betterautomation and accuracy but requires very fast temperature sampling andsignal processing electronics, fast data communication with the reactortransfer mechanism, and very responsive mechanical motion componentssuch as motors and actuators in the reactor transfer mechanism. TiGMCMon the other hand does not require such highly responsive set-up thoughneeds user calibration based on the extent of the temperature offset.The advantageous impact of the temperature-offset feature has beendemonstrated by experimental graphs at FIGS. 12(a) and (b) where thetime of thermal cycling with forty cycles has been shown to reduce toone-fifth. This reduction in time can be further reduced by increasingthe magnitude of the temperature-offsets.

According to an embodiment, a third bath is provided where the bathmedium is maintainable at a medium temperature T_(MEDIUM) for thermalcycling the reactor(s) in three-steps, where the transfer means allowsthe reactor(s) to be in the third bath to attain a predetermined mediumtarget temperature T_(MT). The T_(MT) may be lower than the T_(MEDIUM)for attaining a faster heating rate from the T_(LT) or the T_(MT) may behigher than the T_(MEDIUM) for attaining a faster cooling rate from theT_(HT). The T_(MT) may be maintained same as the T_(MEDIUM).

According to an embodiment, a fourth bath is provided where the bathmedium is maintainable at a temperature T_(AP) to allow an additionalprocess for the reactor(s) before the thermal cycling, the additionalprocess being one from the group consisting: reversetranscription-polymerase chain reaction (RT-PCR), hot start process andisothermal amplification reaction, where the transfer means allows thereactor(s) to attain an additional process target temperature T_(APT),the T_(AP) being same as or higher or lower than the T_(APT). Theadvantage is same as in using the temperature-offset feature. This helpsin integrating the process steps and advantageously allows bath sharingas well with the appropriate temperature setting, thereby saving on footprint and mass of the apparatus.

The bath medium in the first bath may be maintainable above 100 degreesCelsius, to better exploit the advantage of the temperature-offsetfeature with a suitable bath medium that can be heated to such hightemperatures. The bath medium in the second bath may be maintainablebelow room temperature to better exploit the advantage of thetemperature-offset.

According to an embodiment, the transfer means is calibrated to initiatelift-off of the reactor(s) from the bath(s) when the reactor(s) reach afirst lift-off temperature that is lower than the T_(HT) and a secondlift-off temperature that is higher than the T_(LT), in order tocompensate for operational electro-mechanical delays that unwantedlycause over heating or over cooling of the reactor(s).

According to an embodiment the apparatus comprises altering means foraltering temperature in any bath during thermal cycling. This featuresignificantly helps reduce the number of baths in the apparatus therebyreducing the foot print and weight of the apparatus.

The T_(HT) may be in the region 85-99 degree Celsius for denaturation ofthe nucleic acid, and the T_(LT) may be in the region 45-75 degreeCelsius for annealing of primers or probes onto nucleic acid or forprimer extension, the first and the second baths being for thermalcycling the reactor(s) to attain polymerase chain reaction (PCR)amplification or primer extension.

The apparatus may further comprise a fifth bath for a temperaturestabilization step in the thermal profile. The temperature stabilizationstep may be at one of the target temperatures if required for thethermal profile.

The apparatus may further comprise fluorescence imaging means orelectrochemical detection means for analyses of the nucleic acid whenthe reactor(s) is in any of the baths or in air outside the baths.

The apparatus may further comprise a sixth bath wherein the bath mediumis liquid or hot air that is maintainable at 40-80 degree C., whereinthe bath medium and at least a portion of the bath wall is transparentto allow transmission of illumination light from a light source andtransmission of emitted light from the reactor(s).

A first offset between the T_(HIGH) and the T_(HT) may be in the range1-400 degree Celsius and a second offset between the T_(LOW) and theT_(LT) may be in the range 1-100 degree Celsius. Higher the offset,faster is the change of temperature attained by the reactor(s), therebyincreasing the speed of the thermal cycling.

The bath medium in the first bath may be a first liquid added to asecond liquid with a higher boiling point such that the temperature inthe mixture can be maintained at a higher value such as above than 100deg Celsius. without boiling off.

The apparatus may further comprise bath cover(s), the cover(s) openingto allow the reactor(s) to be in the bath(s) and closing after thereactor(s) is/are removed from the bath(s). This feature helps is savingenergy by reducing the heat lost or gained when kept exposed to theambience. Other parts of the apparatus are also prevented from gettingheated up particularly when the T_(HIGH) is set to a value much higherthan the ambience temperature. This also reduces vaporization of thebath medium and contamination of the surrounding parts of the apparatus.

The apparatus comprises bath temperature sensors to monitor temperaturesof the bath mediums and a reactor temperature sensor that is capable ofmoving with the reactor holder during thermal cycling, to monitor thereal time temperature of the reactor(s). The apparatus may furthercomprise a vessel containing a substance to encapsulate the reactortemperature sensor, the vessel and the substance having similarconstruction or heat transfer characteristics to that of the reactor(s)and the reaction material so that the temperature sensed by the reactortemperature sensor is close to the temperature of the reaction materialin the reactors at any instant.

The apparatus may further comprise a seventh bath that can receive thereactor(s) and be progressively heated while conducting melt curveanalysis after the thermal cycling. This helps integrating the processsteps and particularly advantageous in terms of bath sharing.

According to a second aspect, method claims corresponding to theapparatus claims are provided.

The present invention enables the entire process of thermal cycling ofnucleic acid and analysis to be completed in a very short time durationof a few minutes, from bath heating preparation, to reactor thermalcycling and fluorescence signal acquisition. The invention providesscope for bath sharing.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following drawings, same reference numbers generally refer to thesame parts throughout. The drawings are not to scale, instead theemphasis is on describing the concept.

FIG. 1a is a schematic view of an embodiment of a set up for thermalcycling of a reaction material containing nucleic acid as according tothe invention.

FIG. 1b is a perspective view of the reactors being accommodated in anexemplary biochip that is usable for the invention.

FIG. 1c is a cross-sectional view to illustrate the illumination andfluorescence emission detection module with an opaque tubular reactorthat is particularly suited for powder bath medium.

FIG. 2 is an isometric view of an embodiment of the apparatus for theprocess of thermal cycling as at FIG. 1.

FIG. 3 is an isometric view of an embodiment of the reactor array andbaths with optics modules of the apparatus in FIG. 2.

FIG. 4a is an exemplary graphical representation of a typical 2-stepthermal cycling process along with melt curve analysis, according to anembodiment.

FIG. 4b is an exemplary graphical representation of a typical 3-stepthermal cycling process along with melt curve analysis, according to anembodiment.

FIG. 5a is graphical representation of Newton's Law for heating rate.

FIG. 5b is graphical representation of Newton's Law for cooling rate.

FIG. 6(a) is a graphical representation of experimental reactortemperature variations with time during forty cycles of thermal cyclingusing the apparatus previously described and without using thetemperature offset feature.

FIG. 6(b) is a graphical representation of experimental reactortemperature variations with time during forty cycles of thermal cyclingusing the apparatus previously described and using the temperatureoffset feature.

FIGS. 7(a) to 7(e) are graphical representations to describe the conceptof temperature-offset feature according to an embodiment of theinvention.

FIGS. 8(a) and 8(b) are a schematic and elevational cross-sectionalrepresentations to describe the concept of temperature-offset featureaccording to various embodiments of the invention.

FIG. 8(c) is a graphical representation to describe the concept oftemperature-offset feature according to FIGS. 8(a) and (b).

FIG. 9(a) to 9(d) are graphical representations to describe the conceptof temperature-offset feature.

FIGS. 10(a), FIG. 10(b), FIG. 11(a), FIG. 11(b), FIG. 11(c), FIG. 11(d)and FIG. 12 are schematic representations to describe the concept oftemperature-offset feature according to various embodiments of theinvention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The following description presents several preferred embodiments of thepresent invention in sufficient detail such that those skilled in theart can make and use the invention.

FIG. 1a shows a schematic view of an embodiment of a portion of thethermal cycling apparatus for thermal cycling of nucleic acid such asfor PCR, primer extension or other enzymatic reactions. The apparatushas two baths 50 and 51 containing the bath medium 75. Each bath 50 or51 has a bath heater 17 and a bath temperature sensor 39 mounted alongthe bath surface to enable control of the temperature of the bath medium75. According to another embodiment, the bath temperature sensors 39 maybe positioned inside the baths 50, 51. Bath 50 may be suitable for thestep of denaturation and the bath 51 may be suitable for the step ofannealing and/or extension. The cooler 16 is useful when the bath 51needs to be actively cooled to below room temperature. The bath medium75 shown here is liquid, however any other type of fluid or powder orsolid bath medium 75 may also be used. For some embodiments, the bathheater 17 on the low temperature bath 51 is optional, if bath 51 doesnot have to be heated. For the thermal cycling, the reactor 15 isalternately transferred between the baths 50, 51 multiple times. Toenable fast plunging of the reactor 15 into the bath medium 75, slimreactor 15 is preferable such as glass capillaries. The reactor 15 issealed with a sealant or a cap 77 and a portion of the reactor 15 hereinis transparent to allow light to pass through for dye or probeexcitation and fluorescence imaging. A temperature monitoring unit 34 isinstalled on the reactor holder 33 and moves along with the reactor 15between the baths 50, 51. The temperature monitoring unit 34 contains afast response temperature sensor 38 inside. The temperature monitoringunit 34 has a shape similar to that of the reactor 15 and is constructedto have a similar or the same steady state and transient thermalcharacteristics as those of the reactor 15, for the temperature readingand thermal response to be similar or same as those of the reactor 15unless another reactor 15 itself is used for the purpose. For example,the temperature monitoring unit 34 may have the fast responsetemperature sensor 38 inserted into water or oil or a layer of oil overwater 22 and sealed. Although only one reactor 15 is shown, according toother embodiments the reactor holder 33 may accommodate a plurality ofreactors 15. The reactor 15 may be in the form of tube(s) as shown or aswellplate(s) or chip(s) or cartridge(s). The reactor transfer mechanism85 transfers the reactor 15 and the temperature monitoring unit 34 athigh speed among the baths 50 and 51 to expose them alternately to thedifferent temperatures in the baths 50 and 51 as required for thethermal cycling. There are many possible designs of the reactor transfermechanism 85. One such mechanism is comprised of an X stage 86 movingalong an X axis linear guide 87 for the reactor 15 and the temperaturemonitoring unit 34 to reach to a region above the baths 50 and 51, and aZ stage 88 moving along a Z axis linear guide 89 for the reactors 15 tomove them down to enter the bath medium 75 or to be withdrawn from thebath medium 75. Such a transfer mechanism 85 can also consist of arotary arm (not shown) that moves the reactor 15 and the temperaturemonitoring unit 34 in an angular direction along with the Z stage 88moving along the Z axis linear guide 89. The reactors 15 have an openingfor loading and the reaction material 21 and the openings are sealable.The sealant 77 may be made of a silicone rubber or UV cured polymer, hotmelt and/or wax and/or gel which is in solid phase during thermalcycling. The sealing can also be achieved using liquid such as oil,viscous polymer, and gel. The highly viscous liquid can be applied tothe opening and/or top section of the reactors 15, it is able to blockthe vapor generated from the reaction material 21 from leaking out. Thearrangement for the fluorescent imaging may be in any form as in theart. FIG. 1b is a perspective view of the reactors 15 being accommodatedin a card 31 for use with the baths as according to an alternateembodiment for the reactors 15. There is at least one inlet 313 which isin fluid communication with the reactors 15 via a network of channels315. The reaction material 21 to be tested can be loaded into the inlet315 that subsequently flows into the reactors 15. FIG. 1c shows anembodiment particularly where the reactor 15 is made of anon-transparent reactor 15. For the fluorescent imaging, an opticalfiber 309 transmits light from an illumination light source such as anLED (not shown) into the reaction material 21 inside the reactor 15. Theoptical fiber 310 is for light transmission from the reaction material21 to a photodetector (not shown). The sealant or cap 77 holds theoptical fibers 309, 310. Alternately, imaging may also be conducted forat least partially transparent reactor 15 using at least partiallytransparent bath(s) with transparent bath medium 75 as shown in FIG. 3.In some cases imaging may be conducted when the reactor 15 is outsideany bath. Fluorescence imaging of the reactors 15 while the reactors 15are inside the bath medium 75 is important since the reactors 15 mayhave to be maintained at the annealing or extension temperature for aprolonged period of time when multiple images of different wavelengthsare taken for multiplex detection or to acquire fluorescence images ofcontrol genes. At least a portion of the reactor 15 needs to betransparent to allow light to pass through for dye or probe excitationand fluorescence imaging.

FIG. 2 shows an isometric view of the apparatus with the reactortransfer mechanism 85, wherein an optics module 206 carries outfluorescent detection of nucleic acid inside the reactors 15, atemperature controller module 205 controls the bath temperatures, amotion controller 207 controls all motions and a system controller 208controls the system, with data communication and processing, imageprocessing and data analysis. The bath module 204 is shown to comprisefive baths 50, 51, 52, 53, 54 placed next to each other and eachmaintained at a predetermined temperature. In the optics module 206, thereactors 15 are situated in the low temperature bath 51 having air ortransparent liquid as the bath medium 75 in this embodiment. Theadditional processes like reverse transcription-polymerase chainreaction (RT-PCR), hot start process, and isothermal amplificationreaction can also be carried out in any of the baths for thermal cyclingwhich can be set at temperature TA before the thermal cycling and resetto the temp for thermal cycling after the completion of the aboveadditional processes. FIG. 3 shows a blown up illustration of the bathmodule 204 and the optical module 206. The array of reactors 15 with thetemperature monitoring unit 34 are transferred among the baths 50 to 54.In this embodiment, the bath 51 has air or transparent liquid as thebath medium 75 along with a transparent window 25 at the bottom to passthe illumination light and emission light to and from the reactor 15 asshown with the arrows for the ray paths. The bath heaters 17 or cooler16 are connected to the sidewalls of the baths. The apparatus mayfurther comprise a hot air zone (not shown) for placing the reactors 15particularly during imaging. This simplifies the apparatus. There may bean electrical heater or an infrared heater to form the hot air zoneabove a bath or inside a bath. The heater may preferably be installedwith the reactor holder 33 so that only the air in the vicinity of thereactors 15 is heated while the reactor(s) 15 is/are moving between thebaths. This feature saves energy and saves the other parts of theapparatus from getting undesirably heated. The multiple baths 50 to 54may be used as required under the thermal profile for the thermalcycling and for steps before and after the thermal cycling. In athree-step thermal profile for thermal cycling, the reactor 15 isinserted into three baths within each thermal cycle. In between thebaths 50 and 51, the reactor 15 with the monitoring unit 34 may beinserted into a 3rd bath at a medium temperature or positioned in hotair for a period of time required for annealing and or extension. Beforethe thermal cycling, the reactor 15 with the temperature monitoring unit34 may be inserted into a four bath, that is maintained at apredetermined temperature such as for an additional processing beforethe thermal cycling for nucleic acid amplification. The additionalprocess may be from the group consisting reversetranscription-polymerase chain reaction (RT-PCR), hot start process andisothermal amplification reaction. Melt curve analysis may be conductedusing a bath after the thermal cycling.

FIG. 4a is an exemplary time-temperature graphical representations oftypical 2-step thermal cycling process followed by a melt curveanalysis. Only three cycles are shown over the processes of denaturationand annealing employing two baths. After the thermal cycling, thereactors 15 are placed in at least a partially transparent bath medium75 that is progressively heated while melt curve analysis is conducted.The fluorescence signals from the reactors 15 are acquired at multipletemperatures to form a fluorescence-temperature curve for the melt curveanalysis. The speed of thermal cycling is increased by increasing theramp-up and ramp-down of the temperatures of the reactors 15 by thetemperature-offset feature, where the temperature of the bath medium 75in the first bath 50 is maintained at a temperature T_(HIGH) which issignificantly higher than the temperature for denaturation or the targethigh temperature T_(HT) and the temperature of the bath medium 75 in thesecond bath 51 is maintained at a temperature T_(LOW) which issignificantly lower than the temperature for annealing or the target lowtemperature T_(LT). This feature advantageously uses Newton's law ofcooling that states: ‘the rate of change of the temperature of an objectis proportional to the difference between its own temperature and theambient temperature (i.e. the temperature of its surroundings). FIG. 4bis an exemplary time-temperature graphical representations of typical3-step thermal cycling process followed by a melt curve analysis. Onlythree cycles are shown over the processes of denaturation, annealing andextension employing three baths. After the thermal cycling, the meltcurve analysis is conducted as described under FIG. 4a . Herein, thetemperature-offset feature described under FIG. 4a is further extendedfor the third bath 52 where the temperature of the bath medium 75 ismaintained at a temperature T_(MEDIUM) which is significantly higherthan the temperature for extension or T_(MT). The bath medium 75 for themelt curve analysis may be air or transparent liquid and the bath needsto have a transparent window 25, both required to have lowauto-fluorescence.

FIG. 5a illustrates this law, where an object at temperature T_(LT) isshown to heat up to a temperature T_(HT) at a time t1 when the ambienttemperature is at a temperature T_(HIGH), where T_(HIGH)>T_(HT). Thesame object at temperature T_(LT) is shown to heat up to the temperatureT_(HT) at a time t2 when the ambient temperature is at a temperatureT_(HT). As shown, t1<t2, which indicates that the rate of heating isfaster when the ambient temperature is higher. FIG. 5b illustrates thislaw, where an object at temperature T_(HT) is shown to cool down to atemperature T_(LT) at a time t3 when the ambient temperature is at atemperature T_(LOW), where T_(LOW)<T_(LT). The same object attemperature T_(HT) is shown to cool down to the temperature T_(LT) at atime t4 when the ambient temperature is at a temperature T_(LT). Asshown, t3<t4, which indicates that the rate of cooling is faster whenthe ambient temperature is lower. Thus, by maintaining the T_(HIGH) tobe significantly higher than the temperature for denaturation andT_(LOW) to be significantly lower than the temperature for annealing,the rate of change of the temperature of the reactor 15 is significantlyincreased during the thermal cycling, thereby making the thermal cyclingsignificantly faster.

As described under FIGS. 4a and 4b , the concept of using thetemperature-offset may be used at any stage of the thermal cycling usingany number of baths as required. This feature of maintaining atemperature-offset may selectively be used for only heating or onlycooling steps or for both. The liquid bath medium 75 in the hightemperature bath 50 may have a boiling temperature that is higher thanthat of water at 100 degree Celsius to enable faster ramp-up of thereactor temperature.

FIG. 6(a) shows a graphical representation of experimental reactortemperature variations with time during forty cycles of thermal cyclingusing the apparatus previously described and without using thetemperature offset feature by maintaining T_(HIGH)=T_(HT)=95 degreeCelsius and T_(LOW)=T_(LT)=60 degree Celsius. The time taken for fortythermal cycles is 1450 seconds as seen from the time axis. FIG. 6(b)shows a graphical representation of experimental reactor temperaturevariations with time during forty cycles of thermal cycling using theapparatus previously described and using the temperature offset featureby maintaining T_(HIGH)=120 degree Celsius, T_(HT)=95 degree Celsius,T_(LOW)=25 degree Celsius and T_(LT)=60 degree Celsius. The time takenfor forty thermal cycles is 300 seconds as seen from the time axis.Thus, with the temperature-offset feature, the time for thermal cyclinghas been reduced to about one-fifth. This is a very significantimprovement as about 25 minutes has been reduced to 5 minutes. In thiscase slight overshoots are observed in both the high and low temperaturebaths 50, 51 due to delay in the electromechanical system in changingthe baths. Such overshoots can be avoided by calibrating the apparatusto lift-off the reactors 15 from the baths 50, 51 slightly before thetarget temperatures are attained to compensate for the delay.

To enable rapid heating of the reactor 15, according to an embodimentthe liquid bath medium 75 in the high temperature bath 50 issignificantly over-heated to 125 degree Celsius and above the hightarget temperature T_(HT) for DNA denaturation which is typically around95 degree Celsius. A mixture of glycerol and water of a mixing ratio of70:30 or higher can be used to avoid the liquid boiling when above 100degrees Celsius while maintaining a good thermal conductivity of theliquid. To enable rapid cooling of the reactor 15, the liquid in the lowtemperature bath 51 is significantly cooled below the low targettemperature T_(LT) for annealing and/or extension DNA molecules which istypically around 58 degree Celsius. For example, the liquid in the lowtemperature bath 51 may be maintained at 10 or 30 degrees Celsius whilethe room temperature is at 20° C. The reactors 15 contain a reactionmaterial 21 having least one nucleic acid molecule and a reagent foranalyses.

A temperature guided motion controlling means (TeGMCM) or a time guidedmotion controlling means (TiGMCM) (not shown) is employed in theapparatus for allowing the reactors 15 to remain in the baths 50 to 54until substantially the corresponding target temperature is attained,irrespective of the temperatures of the corresponding baths. TheTeGMCM/TiGMCM may be fed with advance signals when the reactor(s) 15 areabout to reach the target temperatures as sensed by the temperaturemonitoring unit 34 in order to avoid over heating or over cooling of thereactors 15 due to any operational electro-mechanical delay in removingthe reactor 15 from the bath. This helps to maintaining better accuracyof the predetermined target temperatures attained by the reactors 15.Advantageously, with the TeGMCM/TiGMCM, the required number of hightemperature and low temperature baths can be reduced in the apparatus byallowing the reactor 15 to attain multiple levels of temperature basedon the time for which it is allowed to remain in the bath. Forstabilization at any temperature level however dedicated bath at thattemperature is required.

The above temperature-offsets with the over-heating and under-heatingmethods make the temperature ramp-up/ramp-down for the reactor 15faster. The theory is explained in this section containing Eq (1-7). Onetype of our reactor 15 in operation comprises a tube containing thereaction material. The tube and the reaction material 21 have differentthermal and other material properties. To illustrate the heat transfercharacteristics of the reaction material 21 loaded reactor 15 submergedin bath medium 75 during thermal cycling, we approximate the submergedreactor 15 loaded with the reaction material 21 as a cylinder made of ahomogeneous material having the outer surface area A_(s), the radius R,the length L, the volume of the submerged cylinder V, the heat capacityc_(p). In order to estimate the time of heating of the cylindersubmerged in the high temperature bath from the T_(LT) when the cylinderenters the high temperature bath to the T_(HT) during thermal cycling,we determine the average rate of heat transfer {dot over (Q)}_(avg) fromNewton's Law of Cooling by using the average surface temperatureT_(s, avg) of the cylinder [Reference 1: Y. A. Cengel and A. J. Ghajar,Heat and Mass Transfer: Fundamentals and Applications, Fifth Edition InSI Units by McGraw-Hill Education, 2015]. That is,

{dot over (Q)} _(avg) =−hA _(s)(T _(s,avg) −T _(HIGH)),  Eq (1)

where T_(HIGH) is the temperature of bath medium in the high temperaturebath, T_(s, avg)=(T_(LT)+T_(HT))/2. Under the over-heating scheme in thehigh temperature bath, T_(HIGH)>T_(HT). Note: an example of the T_(LT)is the annealing temperature in PCR, and an example of the T_(HT) is thedenaturation temperature in PCR.Next, we determine the total heat transferred from the cylinder[Reference 1], which is simply the change in energy of the cylinder asit heats from T_(LT) to T_(HT):

Q _(total) =Vc _(p)(T _(HT) −T _(LT)),  Eq (2)

In this calculation, we assumed that the entire cylinder is at uniformtemperature over the domain of the cylinder. With this assumption, thetime of heating the cylinder from T_(LT) to T_(HT), Δt, is determined tobe

$\begin{matrix}{{\Delta \; t} = {\frac{Q_{total}}{{\overset{.}{Q}}_{avg}} = \frac{V\; {c_{p}( {T_{LT} - T_{HT}} )}}{h\; {A_{5}\lbrack {{0.5( {T_{HT} + T_{LT}} )} - T_{HIGH}} \rbrack}}}} & {{Eq}\mspace{14mu} (3)}\end{matrix}$

Because of the assumption made above, Eq (3) does not yield accuratetemperature value, but it reveals the factors of influencing the time ofheating up the cylinder.The heat transfer coefficient h in Eq (3) is related to the relativevelocity between the heating medium and the reactor, which can beobtained from the following analysis of convective heat transfer in anexternal flow across a cylinder [Reference 1]:The average Nusselt number for flow across the cylinder can be expressedcompactly as

Nu=CR _(e) ^(m) P _(r) ^(n),  Eq (7-37) of Reference 1

where the constant C, m, and n are related to the Reynolds number Rewhich is defined as

$R_{e} = \frac{\rho \; {vD}}{\mu}$

where D is the diameter of the cylinder, ρ is the density of the liquidin bath, μ is the dynamic viscosity of the liquid in bath, and v is therelative velocity between the heating medium and the reactor.For example, if R_(e) is in the range of 40-4,000, Eq (7-37) can berewritten as

Nu=0.683R _(e) ^(0.466) P _(r) ^(1/3)  Eq (4),

which is as shown in Table 7-1 in Reference 1.

Since

${{Nu} = \frac{hD}{k}},$

where h is the heat transfer coefficient on the cylinder surface, D isthe diameter of the cylinder, and k is the thermal conductivity ofliquid in the bath, Eq (4) can be rewritten as

$\begin{matrix}{{h = {0.683\; \frac{k}{D}( \frac{\rho \; D}{\mu} )^{0.466}{P_{r}^{1/3}(v)}^{0.466}}},} & {{Eq}\mspace{14mu} (5)}\end{matrix}$

Inserting Eq (5) into Eq (3), we obtain

$\quad\begin{matrix}\begin{matrix}{{\Delta \; t} = \frac{Q_{total}}{{\overset{.}{Q}}_{avg}}} \\{= \frac{V\; {c_{p}( {T_{HT} - T_{LT}} )}}{{A_{s}\lbrack {T_{HIGH} - {0.5( {T_{HT} + T_{LT}} )}} \rbrack}0.683\; \frac{k}{D}( \frac{\rho \; D}{\mu} )^{0.466}P_{r}^{1/3}v^{0.466}}}\end{matrix} & {{Eq}\mspace{14mu} (6)}\end{matrix}$

Eq (6) shows that advantage of implementing the over-heating strategy;that is, the higher the over-heated bath temperature T_(H), the shorterthe time Δt of heating the cylinder from T_(LT) to T_(HT).Experimentally, FIG. 5a shows that when the over-heated bath temperatureT_(HIGH) is raised to 115° C. from a temperature of 95° C., the time t1for the reactor 15 to reach the 95° C. (T_(HT)) from the 50° C. (T_(LT))is much shorter than the time t2 for the reactor 15 to reach 95° C.(T_(HT)) when the bath medium 75 is set at 95° C.

Similarly, the time of cooling the cylinder from T_(HT) to T_(LT), Δt,is determined to be

$\begin{matrix}\begin{matrix}{{\Delta \; t} = \frac{Q_{total}}{{\overset{.}{Q}}_{avg}}} \\{= \frac{V\; {c_{p}( {T_{HT} - T_{LT}} )}}{{A_{s}\lbrack {{0.5( {T_{HT} + T_{LT}} )} - T_{LOW}} \rbrack}0.683\; \frac{k}{D}( \frac{\rho \; D}{\mu} )^{0.466}P_{r}^{1/3}v^{0.466}}}\end{matrix} & {{Eq}\mspace{14mu} (7)}\end{matrix}$

Eq (7) shows that advantage of implementing the under-heating strategy;that is, the lower the under-heated bath temperature T_(LOW), theshorter the time Δt of cooling the cylinder from T_(HT) to T_(LT).Experimentally, FIG. 5b shows that when the under-heated bathtemperature T_(LOW) is reduced to 20° C. from a temperature of 50° C.,the time t3 for the reactor 15 to reach 50° C. (T_(LT)) from 95° C.(T_(HT)) is much shorter than the time t4 for the reactor 15 to reachthe 50° C. (T_(LT)) when the bath is set at the 50° C.

Various exemplary methods for conducting rapid thermal cycling andnucleic acid processing using the apparatus described above aredescribed as follows. When performing thermal cycling by transferringthe reactors 15 alternately between a high temperature bath 50 and a lowtemperature bath 51, FIG. 7(a) shows the reactor temperature variationcurve 200 (dotted curve) in a preferred embodiment of a method in thisinvention using the over-heating feature in the high temperature bath 50and the temperature variation curve 300 (dashed curve) in anotherembodiment without using the overheating method in the high temperaturebath 50. Thus, during thermal cycling the temperature ramping up rate ofthe reactor 15 is faster for the curve 200 than for the curve 300.Herein, the high temperature bath 50 is set at a temperature T_(HIGH)that is higher than the T_(HT) as required for the denaturationtemperature and the low temperature bath 51 is set at the T_(LT) that issuitable for annealing and/or extension. After the baths are heated tothe respective bath temperatures T_(HIGH) and T_(LT), the reactortransfer mechanism 85 transfers the reactors 15 to the high temperaturebath 50 to initiate the thermal cycling for the PCR or otheramplification reaction with or without a hot start step which may or maynot require a separate bath of a suitable temperature for the hot startstep. Once the reactor temperature corresponding to the curves 200 or300 reaches the T_(HT) in the high temperature bath 50, the reactortransfer mechanism 85 pulls out the reactors 15 and transfers them tothe low temperature bath 51 for the reactors 15 to reach T_(LT), andsubsequently transfers the reactors 15 back to the high temperature bath50 for thermal cycling. In this embodiment, the reactor 15 is allowed tostay in the bath 51 for a period of time, called residence time t_(L) asrequired for nucleic acid amplification. Once the residence time t_(L)is reached in the bath 51, the reactor transfer mechanism 85 lifts upthe reactors 15 out of the bath 51 to transfer to the bath 50 tocontinue the thermal cycling. Similar residence time may also be appliedin the high temperature bath 50 (not shown). In one embodiment, theresidence time t_(L) can be measured by the reactor temperaturemonitoring unit 34 in the apparatus that moves together with the reactorholder 33 as shown in FIG. 1a . However, this method may be costly toimplement. When the reactors 15 plunge into a bath, the reactortemperature curves 200 or 300 can change at an extremely high rate, suchas 40 degree Celsius to 90 degree Celsius per second. To controlprecisely the timing of lifting the reactor 15 by the reactor transfermechanism 85, it requires a very fast temperature sampling and signalprocessing electronics, fast data communication with the reactortransfer mechanism 85, and very responsive mechanical motion componentssuch as motors and actuators in the reactor transfer mechanism 85. Butpractically, such a fast electronic device may not be available or istoo costly to a particular entity making the present apparatus. Inanother embodiment, the residence time t_(L) is counted from the momentthe reactors 15 are immersed into a bath. For example, the reactor 15 isimmersed in a bath for 4 seconds before being moved out of the bath,given an assumption that the reactors 15 have reached the targettemperature T_(LT) or its vicinity within 1 second after immersion andstayed at the target temperature T_(LT) or its vicinity for 3 seconds.This method is important since it can be implemented at low cost,without a need for any device capable of high speed temperaturesampling, processing and data communication. Similar ultra-rapid heatingand cooling is observed also for plastic and metal reactors 15 ofvarious shapes. For plastic reactors 15, we particularly use a T_(LT)substantially below the room temperature using active cooling devicessuch as thermoelectrical coolers. The apparatus may allow user selectionfor the conditions T_(HIGH)=T_(HT) or T_(LOW)=T_(LT) if desired.

FIG. 7(b) describes a method of conducting thermal cycling usingover-heating in the high temperature bath 50 and under-heating in thelow temperature bath 51. The temperature T_(LOW) in the low temperaturebath 51 is set below the T_(LT) so that the heat transfer between thereactor 15 and the bath medium 75 in the low temperature bath 51 is muchstronger, leading to a much more rapid cooling of the reactor 15. Whenthe reactor temperature reaches the T_(LT), the reactor transfermechanism 85 lifts up the reactors 15 immediately and transfers them tothe high temperature bath 50 to continue thermal cycling. FIG. 7(c)further describes a thermal cycling method with the reactor temperatureovershooting above the T_(HT) and below the T_(LT). This method enablesthe reactors 15 to stay in the vicinity of the T_(HT) for a longerperiod of time t_(H) for denaturation and/or to stay in the vicinity ofthe T_(LT) for a longer period of time t_(L) for annealing andextension. In another embodiment shown in FIG. 7(d), after the reactors15 reach a temperature in a vicinity of the T_(LT), the reactors 15 aremoved out of the low temperature bath 51 into a zone of air to maintainthe reactor temperature 13 in the vicinity or at T_(LT) for a timeperiod required for nucleic acid analysis such as annealing and/orextension in PCR, and then the reactors 15 are moved to the hightemperature bath 50 to continue thermal cycling. Air in the air zone canbe heated and the heated air zone can be established near the lowtemperature bath 51 using an infrared heater, a fan blowing hot air, anelectrical resistance wire heater, or be a simple air zone at a roomtemperature. The heated air zone can also be created above the bathmedium 75 within the either high temperature bath 50 or low temperaturebath 51 to utilize the confined space in the baths 50, 51 for bettermaintenance of reactor temperature 13. The heated air zone can also becreated above the bath medium 75 at the opening of the either hightemperature bath 50 or low temperature bath 51. The same strategy canalso be applied to the high temperature bath 50 for maintaining theT_(HT) for a prescribed period of time.

In another embodiment shown in FIG. 7(e), after the reactors 15 reachbelow the T_(LT) in the low temperature bath 51, the reactor transfermechanism 85 moves the reactors 15 out of the bath 51 into a heated airzone to heat the reactors 15 above the T_(LT), and subsequently movesthe reactors 15 into the low temperature bath 51 to cool the reactors 15below the T_(LT), and repeats this operation as many times as requiredto generate an oscillatory temperature variation around the T_(LT) forannealing and primer extension. The same strategy can also be applied tothe high temperature bath 50 by using a cooled air zone for maintainingthe T_(HT) for a prescribed period of time.

FIG. 8(a) shows another embodiment of thermal cycling by rapidly coolingthe reactors 15 using one under-heating bath 61 which is maintained at atemperature 35 below the T_(LT). The reactor 15 is then moved to the lowtemperature bath 51 to maintain the reactor temperature 13 in thevicinity of the T_(LT). During thermal cycling along reactor transferpath 56, the reactors 15 leave the high temperature bath 50 which couldbe under an over-heating condition and move into the under-heating bath61 to rapidly reach to the vicinity of the T_(LT), and then move intothe low temperature bath 51 to sustain at the T_(LT) for a requiredperiod of time and carry out fluorescent imaging of the reactors 15 forconducting real-time quantitative PCR, before moving the reactors 15back to the high temperature bath 50 for thermal cycling. The abovedescribed fluorescent imaging of the reactors 15 at every cycle of thethermal cycling may not be necessary for every application. For someapplications, the fluorescent imaging of the reactors 15 can be carriedout at every few cycles in order to reduce the total time of nucleicacid analysis. The fluorescent imaging of the reactors 15 can be carriedout at the end of the thermal cycling for conducting end-point PCRdetection. FIG. 8 (b) shows the schematics of the high temperature bath50, under-heating bath 61, and the low temperature bath 51 in thisembodiment. The under-heating bath 61 can also be cooled to below a roomtemperature. The lower the temperature of the under-heating bath 61, thefaster the cooling of the reactors 15 to the vicinity of the T_(LT).Before the reactors 15 are moved out of the under-heating bath 61, thereactor temperature 13 can be same or lower or higher than the T_(LT).It may be difficult to cool the reactors 15 exactly to the T_(LT) in theunder-heating bath 61 before transferring to the low temperature bath51. Such difficulties include availability of a temperature controllercapable of high sampling rate from temperature sensor, fast temperaturesignal processing, fast temperature data transfer to temperaturecontroller or a signal processing controller through a communicationport, a time lag for the motion mechanism to move the reactors 15 out ofthe low temperature bath 51, and the like. Therefore, it is morerealistic to have the reactors 15 to be cooled to the vicinity of theT_(LT) with a temperature range of such vicinity to be 0.5-5° C. belowor above the T_(LT) in the under-heating bath 61 before transferring tothe low temperature bath 51. To speed up the temperature change to reachthe T_(LT) after moving the reactors 15 from the under-heating bath 61to the low temperature bath 51, the low temperature bath 51 can be setat a temperature different from the T_(LT). For example, if the reactors15 in the under-heating bath 61 of temperature 35 is cooled to below theT_(LT), the temperature 14 in the low temperature bath 51 can be sethigher than the T_(LT), as shown in FIG. 8(c). Similarly, if thereactors 15 in the under-heating bath 61 is cooled to above the T_(LT),the temperature 14 in the low temperature bath 51 can be set lower thanthe T_(LT). Specifically, the temperature in the low temperature bath 51can be in the vicinity of the T_(LT). Choice of bath medium temperaturein the under-heating bath 61 can vary according the heat transfercharacteristics of the reactors 15. For example, for the reactors 15made of glass or metal materials, the temperature 35 of theunder-heating bath 61 can be set at room temperature or in its vicinity,whereas for the reactors 15 made of plastics, the temperature 35 of theunder-heating bath 61 can be set substantially lower than a roomtemperature, or at a room temperature. The above temperature setting isalso dependent on the volume of the reactors 15 and wall thickness ofthe reactors 15. For large reactors 15 and the reactors 15 made ofplastics, the under-heating bath 61 can be actively cooled bythermoelectric coolers or a recirculation liquid cooling system to reacha very low bath temperature such as slightly above 0° C. to 20° C. Withthe use of anti-freezing agent or use of salt, sub-zero liquid can beused in the under-heating bath 61 to further enhance the reactor coolingrate. The above intense under-heating or cooling means not only cool theplastic reactors 15 rapidly, but also cool the glass or metal orplastic-metal reactors 15 rapidly. Moreover, when the thermal cyclingscheme shown in FIG. 7 (c) incorporates an over-heating bath for rapidheating to the T_(HT), the entire thermal cycling process can be furthershortened. Furthermore, to shorten the time after transferring thereactors 15 from the under-heating bath 61 to the low temperature bath51, the low temperature bath 51 can be set at a temperature higher thanthe T_(LT), which allows the reactor temperature rise above the T_(LT)to achieve a required period of time in the vicinity of the T_(LT), orcarry out the cyclic transfer of the reactors 15 between theunder-heating bath 61 and the low temperature bath 51, similar to theprocess shown in FIG. 6(e), to achieve a required period of time in thevicinity of the T_(LT). In the above scheme, the under-heating bath 61can be maintained at a room temperature or below a room temperature orabove a room temperature. The same strategy of maintaining the reactortemperature 13 at a target temperature for a period of time shown inFIGS. 7(a, b, c) can be applied to the high temperature bath 50 formaintaining reactor temperature 13 at the T_(HT) for a period of time.

FIG. 9(a) describes an embodiment with deployment of stabilization baths(a 3-step PCR). The reactor temperature 13 is ramping up 82 in the hightemperature bath 50 when set at temperature T_(HIGH). The reactortemperature 13 is stabilizing 92 is when the reactor 15 is in thestabilization bath at T_(HT). The reactor temperature 83 is ramping downin the low temperature bath 51 set at temperature TL. The reactortemperature 13 is stabilizing 93 when the reactor 15 is in thestabilization bath at T_(LT). The reactor temperature 13 is ramping up84 to the medium temperature at T_(MEDIUM). The reactor temperature 94is when the reactor 15 is in the stabilization bath for the reactor 15to maintain at the T_(M)r. FIG. 9(b) describes another embodiment of a3-step PCR as in FIG. 8(a) without deployment of stabilization baths atT_(HT) and T_(LT). FIG. 9(c) describes another embodiment of a 3-stepPCR with deployment of stabilization baths where the bath medium 75 indifferent baths are different. For example, the bath medium 75 in thehigh temperature bath 50 can be thermally conductive particles or aliquid having high evaporative temperature such as over 100 degreeCelsius and the bath medium 75 in the low temperature bath 51 is purewater. The reactor temperature 13 is ramping up 82 in the hightemperature bath 50 when set at temperature T_(HIGH) and containingmetal powder or liquid. The reactor temperature 13 is ramping down 83 inthe low temperature bath 51 set at temperature T_(LOW) and containingmetal powder or liquid. The reactor temperature 13 is ramping up 84 inthe medium temperature or high temperature bath set at temperatureT_(MEDIUM), containing metal powder or liquid. The reactor temperature13 is when the reactor 15 is stabilizing 94 in the stabilization bathfor the reactor 15 to maintain at the medium target temperature T_(MT),with the stabilization bath containing metal powder or liquid or heatedair. The reactor temperature 13 is achieved when the reactor 15 isstabilizing 95 in another stabilization bath containing heated air or anair zone outside the stabilization bath for taking at least onefluorescent image of the reactor 15. FIG. 9(d) illustrates the method ofFIG. 8(c) but without the bath set at temperature T_(MEDIUM) and thestabilizings 94, 95 being conducted at T_(LT)

FIG. 10(a) shows another embodiment in which an over-heating bath 60 isadded to the path 56 for transferring the reactors 15 during thermalcycling to speed up the temperature ramping to T_(HT) in theover-heating bath 60. During thermal cycling, the reactors 15 leave thelow temperature bath 51 and move into the over-heating bath 60 torapidly reach to the vicinity of T_(HT), and then move into the hightemperature bath 50 to sustain in the vicinity of the T_(HT) for arequired period of time before moving back to the low temperature bath51 and optionally via the under-heating bath 61 before moving to the lowtemperature bath 51. The higher the temperature of the over-heating bath60, the faster is the heating of the reactors 15 to the vicinity ofT_(HT). Preferably, the temperature in the over-heating bath 60 isbetween 100 degree Celsius to 135 degree Celsius. Similar to the bathsetup and temperature control described in FIGS. 7a-c , the hightemperature bath 50 can be set at a temperature different from theT_(HT). Specifically, the temperature in the high temperature bath 50can be in the vicinity or outside of T_(HT). FIG. 10(b) shows anotherembodiment in which the high temperature bath 50 is removed fromembodiment in FIG. 9(a). During thermal cycling in this embodiment, thereactors 15 leave the low temperature bath 51 and move into theover-heating bath 60 to rapidly reach to the vicinity of the T_(HT), andthen move back to the low temperature bath 51 and optionally via theunder-heating bath 61 before moving to the low temperature bath 51.

FIG. 11(a) shows a method to perform biological or biochemical reactionsinvolving 3 temperatures, including the 3-step PCR, in which threeheating baths are assembled in a path 56 for transferring the reactors15 for thermal cycling. The three baths include a high temperature bath50 for denaturation, a mid temperature bath 52 for primer extension, anda low temperature bath 51 for annealing. FIG. 11(b) shows thatadditional under-heating bath 61 is optionally added into the thermalcycling path 56 before the low temperature bath 51, and an over-heatingbath 60 is optionally added into the thermal cycling path 56 each beforethe mid temperature bath 52 and the high temperature bath 50,respectively. FIG. 11(c) shows an embodiment modified from embodimentshown in FIG. 10(b), in which the over-heating bath 60 added before themid temperature bath 52 is removed from the thermal cycling path 56.During thermal cycling in this embodiment, the reactors 15 leave the lowtemperature bath 51 and enter the over-heating bath 60 set for hightemperature bath 50 or enter the high temperature bath 50, in dashedline to rapidly reach to the vicinity of the mid target temperature 5 asshown in FIG. 8(a), and then move to the mid temperature bath 52 tostabilize at the mid target temperature 5 for a required period of timefor extension. Once the extension is completed, the reactors 15 move tothe over-heating bath 60 to rapidly reach to the vicinity of the T_(HT),and then move to high temperature bath 50 for a required period of timefor denaturation, before moving back to the low temperature bath 51 oroptionally via the under-heating bath 61 before moving to the lowtemperature bath 51. FIG. 11(d) shows an embodiment modified fromembodiment shown in FIG. 10(c), in which the low temperature bath 51 andthe high temperature bath 50 are moved. During thermal cycling in thisembodiment, the reactors 15 enter the under-heating bath 61, and oncethe reactors 15 reach the T_(LT) as shown in FIG. 8(b), the reactorsleave the under-heating bath 61 and enter the over-heating bath 60 setfor high temperature bath 50 to rapidly reach to the vicinity of the midtarget temperature 5 as shown in FIG. 8(b), and then move to the midtemperature bath 52 to stabilize at the mid target temperature 5 for arequired period of time for extension. Once the extension is completed,the reactors 15 move to the over-heating bath 60 to rapidly reach to thevicinity of the T_(HT), before moving back to the under-heating bath 61.In the embodiment described in FIGS. 10 to 11, the reactors 15 can beimaged for fluorescent detection inside one of the bath 51, 52, 60, and61, if the bath medium 75 inside is transparent to light transmissionand has low auto-fluorescence, and such bath medium 75 can be water,water-glycerol mixture, oil, and heated or un-heated air. If the bathmedium 75 inside bath 51, 52, 60, and 61 are opaque to lighttransmission or has high auto-fluorescence, the reactors 15 can be movedto a zone of heated or un-heated air outside the baths or above the bathmedium inside the bath for fluorescent imaging. The methods of rapidlytransferring the reactors 15 among the heat baths set at differenttemperatures for nucleic acid samples can be incorporated withadditional at least one pre-process like reverse transcription and/orisothermal amplification and/or primer extension. FIG. 12 shows a methodto perform biological or biochemical reactions involving at least onepre-heating bath 53 before starting PCR. The at least one pre-heatingbath 53 can be set at a temperature suitable for reverse transcriptionand/or isothermal amplification and/or primer extension. After exposingthe reactors 15 to the pre-heating bath 53, the reactors are transferredto other bath for isothermal DNA process step and/or PCR thermalcycling. In the embodiment shown, many baths are optional depending onnucleic acid processing requirement, and the optional baths includingthe over-heating baths 60, the under-heating bath 61, and the midtemperature bath 52. In another embodiment, no over-target heatingmethod, i.e., neither over-heating nor under-heating, is used. In thisembodiment, the baths are set at the target temperature required fornucleic acid analysis or processing. In this embodiment, one or morethan one of the following means are used:

-   1) the reactor moves at high speed in a reciprocating manner inside    at least one bath,-   2) reactor transfer mechanism 85 transfers the reactor from one bath    to another in less than 4 seconds, and preferably less than 1    second,-   4) the baths have a high-aspect-ratio geometry with bath heaters 17    being deployed on the bath surfaces forming a shorter bath    dimensions, and-   4) the bath 51 having a lower temperature has an optically    transparent window 27 for fluorescent imaging of liquid sample    inside the reactor 15 situated inside the bath 51.

Small reactors 15 with narrow internal cavities such as a small boreglass capillary are difficult for loading sample and reagent liquid by anormal pipette since air can be trapped underneath the liquid inside thereactor cavity. The following means can be used to load the liquid intosuch narrow cavities: 1) centrifuging the liquid dispensed to theentrance of the reactor cavity, 2) inserting a tube thinner than theinternal passage of the cavity down to the bottom of the cavity, 3)using a vacuum to remove air inside the cavity before loading theliquid, 4) using a pre-vacuumed cavity to load the liquid, and 5) usinga cavity with at least one vent when loading the liquid, and sealing thevent. After loading the liquid, the loading ports are to be sealedbefore thermal cycling starts.

Different bath may contain different bath medium 75 for specificadvantages as desired. The reactors 15 may be made up of plastics,elastomer, glass, metal, ceramic and their combinations, in which theplastics include polypropylene and polycarbonate. The glass reactor 15can be made in a form of a glass capillary of small diameters such as0.1 mm-3 mm OD and 0.02 mm-2 mm ID, and the metal can be aluminum inform of thin film, thin cavity, and capillary. Reactor materials can bemade from non-biological active substances with chemical or biologicalstability. At least a portion of the reactor 15 is preferred to betransparent. In another embodiment, the reactors 15 can be in a form ofa reactor array chip or a microfluidic reactor chip or arrayed chip. Forexample, the reactors 15 can be in a form of wells or channels of asubstrate plate and optionally covered with a solid layer of material toform closed reaction chambers, in which the reaction material 21 issituated.

The reaction material 21 in all the reactors 15 in the reactor holder 33may not be identical. Simultaneous PCR can be advantageously conductedfor different materials if the bath temperatures are suitable. At leastpart of the reactor wall may be made of metal sheet of thickness 1 μm-2mm. This feature enhances the rate of heat transfer between the bath andthe reaction material 21. At least part of the reactor wall may be madeof plastic or glass sheet of thickness 0.5 μm-500 μm. At least a part ofthe reactor wall is made of transparent material so as to enable theimaging and detection process.

The invention is equally applicable for a single reactor 15 or multiplereactors 15 in the reactor holder 33. The term ‘liquid’ used in theabove description is a general term for the heating medium. For thisinvention, the heating medium may be in different forms, such as liquid,water, water mixed with solvent or other chemical fluid or other solidparticles, solid particles, metal particles, copper particles andpowders.

In order to further explain and clearly understand the invention, someexamples and illustration below are combined to further instruct theinvention, especially of the methods to use the apparatus to carry outthe sample testing.

Example 1.1

A method for nucleic acid analysis, comprising the following steps:adding at least one kind of reaction material 21 into at least onereactor 15, sealing the reactor 15, amplifying nucleic acid, and duringthe nucleic acid amplification, employing over-heating and/orunder-heating method, which describes that the temperature of at leastone heating bath is higher or lower than the target temperature requiredfor nucleic acid amplification. The reactor 15 is alternately arrangedin at least two heating baths with different temperatures for thermalcycle when the nucleic acid is amplified. When the temperature of thereactor 15 comes close or equal to the target temperature required forthe nucleic acid amplification, the reactor 15 is moved from the heatingbath where the reactor 15 is in to another heating bath quickly. Whenthe over-heating and/or under-heating method adopts the three-stepmethod for nucleic acid amplification, the temperature of the heatingbath I is set to higher than the target temperature required forpre-denaturation and denaturation, and the temperature of the heatingbath II is set to lower than the target temperature required fordenaturation, and the temperature of the heating bath III is set tohigher than the target temperature required for extension, and thethermal cycle of the reactor is alternately carried out according to anorder of the heating bath I, the heating bath II, the heating bath III,the heating bath I.

Example 1.2

The difference between the present example and example 1.1 is that whenthe temperature of the reactor 15 achieves the target temperaturerequired for the nucleic acid amplification, the reactor 15 is movedfrom the heating bath I and transferred to the heating bath II quickly.

Example 1.3

The difference between the present example and example 1.1 is that whenthe temperature of the reactor 15 exceeds the target temperaturerequired for the nucleic acid amplification, the reactor 15 is quicklymoved from the heating bath III in to the heating bath IV, and thetemperature of the heating bath IV is set to the target temperaturerequired for extension.

Example 2.1

The difference between the present example and example 1.1 is that italso comprises reverse transcription (Reverse Transcription-PolymeraseChain Reaction) before the nucleic acid amplification.

Example 2.2

The difference between the present example and example 2.1 is that thereverse transcription is carried out prior to the thermal cycle of thenucleic acid amplification or simultaneously.

Example 2.3

The difference between the present example and example 2.2 is that thereverse transcription is carried out prior to the nucleic acidamplification, which needs to add a heating bath or to set thetemperature of the heating bath to the target temperature required forthe reverse transcription and then to set the temperature of heatingbath to the target temperature required for nucleic acid amplification.

Example 3.1

The difference between the present example and example 1.1 is that whenthe overtemperature method adopts the three-step method for nucleic acidamplification, the temperature of the 1st heating bath is set to higherthan the target temperature required for pre-denaturation anddenaturation, the temperature of the heating bath II is set to thetarget temperature required for pre-denaturation and denaturation, andthe temperature of the heating bath III is set to lower than the targettemperature required for annealing, and the temperature of the heatingbath IV is set to the target temperature required for annealing, and thetemperature of the heating bath V is set to the target temperaturerequired for extension, and the thermal cycle of the reactor 15 isalternately carried out among the heating baths with differenttemperatures according to an order of the heating bath I, the heatingbath II, the heating bath III, the heating bath IV, the heating bath V,the heating bath I, and so on.

Example 3.2

The difference between the present example and example 1.1 is that theovertemperature method is used to carry out the polymerase chainreaction, and the reactor 15 is alternately placed in the heating bathswith different temperatures. The heating baths is respectively heatingbath I, heating bath II, heating bath III, heating bath IV, heating bathV, heating bath VI. A selected temperature in the heating bath I is105-135° C. A selected temperature of the heating bath II is 95° C. Aselected temperature of the heating bath III is 10-40° C. A selectedtemperature of the heating bath IV is 50° C. A selected temperature ofthe heating bath V is 82-112° C. A selected temperature of the heatingbath VI is 72° C. The selected temperature of the heating bath I ishigher than the target temperature required for pre-denaturation anddenaturation, the selected temperature of the heating bath III is lowerthan the target temperature required for annealing, and the selectedtemperature of the heating bath V is higher than the target temperaturerequired for extension. The temperature of the reactor in the heatingbath I will achieve, come close to or exceed the target temperature 95°C. for a few seconds, and the reactor is moved from the heating bath Ito the heating bath II quickly. The temperature of the reaction systemin the heating bath III will achieve, come close to or exceed the targettemperature 50° C. for a few seconds, and then the reactor is moved tothe heating bath IV quickly. The temperature of the reaction system inthe heating bath V will achieve, come close to or exceed the targettemperature 72° C. for a few seconds, and then the reactor 15 is movedto the heating bath VI quickly. Then the reactor 15 is moved in turn tothe heating bath I, the heating bath II, the heating bath III, theheating bath IV, the heating bath V, heating bath VI, the heating bath Iaccording to motion track A, and with such 35 cycles, the whole processneeds just a few minutes. Of course, the transfer here can be artificialone, it also can be mechanized done using the reactor transfer mechanism85 shown in FIG. 1. No matter which kind, compared with the ordinaryPCR, this kind of method is time-saving, high-speed, high-efficiency andis suitable for the detection in emergence. If using PCR instrument, theprocess is divided into 95° C. pre-denaturation 4 min, 95° C.denaturation 1 min, 50° C. annealing 1 min, 72° C. extension 1.5 min,72° C. extension 5 min, 95° C. denaturation 1 min, 50° C. annealing 1min, 72° C. extension 1.5 min for 35 cycles, and the last is kept in 4°C. The whole process takes nearly two hours and is slow-speed andlow-efficiency.

Example 3.3

The difference between the present example and example 1.1 is that, inany of the above schemes, preferably, when the overtemperature methodadopts the two-step method for nucleic acid amplification, thetemperature of the heating bath I is set to higher than the targettemperature required for pre-denaturation and denaturation and thetemperature of the heating bath II is set to lower than the targettemperature required for annealing or extension.

Example 3.4

The difference between the present example and example 3.3 is that whenthe over-temperature method adopts the two-step method for nucleic acidamplification, the temperature of the heating bath I is set to higherthan the target temperature required for pre-denaturation anddenaturation, the temperature of the heating bath II is set to lowerthan the target temperature required for annealing, the temperature ofthe heating bath III is set to the target temperature required forannealing, and the reactor 15 transfers from the heating bath I to theheating bath II and transfers quickly to the heating bath III as thetemperature lower than the target temperature required for annealing andtransfers to the heating bath I after a certain time for alternatecirculation, and the thermal cycle step of nucleic acid amplification isthe heating bath I, the heating bath II, the heating bath III, then theheating bath II.

From the foregoing description it will be understood by those skilled inthe art that many variations or modifications in details of design,construction and operation may be made without departing from thepresent invention as defined in the claims.

1. An apparatus for thermal processing of nucleic acid in a thermalprofile, the apparatus employing a reactor holder for holding a reactor,wherein the reactor is configured to accommodate a reaction materialcontaining the nucleic acid, the apparatus comprising: two bathsincluding a first bath and a second bath; wherein bath mediums in thetwo baths are configured to be maintainable at two differenttemperatures T_(HIGH) and T_(LOW), and a transfer means configured forkeeping the reactor to be in the two baths in a plurality of thermalcycles to alternately attain: a predetermined high target temperatureT_(HT), and a predetermined low target temperature T_(LT), while theapparatus adapts to a temperature-offset feature defined by at least onecondition selected from the group consisting of a) the T_(HT) is lowerthan the T_(HIGH), and b) the T_(LT) is higher than the T_(LOW); thetransfer means being operable by at least one mode selected from thegroup consisting of: a temperature guided motion controlling means(TeGMCM) operable based on a real-time temperature as sensed by areactor temperature sensor during thermal cycling, and a time guidedmotion controlling means (TiGMCM) operable based on time-periods forwhich the reactor are allowed to be in the two baths; the bath mediumsin the two baths are in gaseous form, liquid form, solid form, powderform or combination thereof.
 2. The apparatus according to claim 1,wherein, the TiGMCM is configured to be calibrated for the time-periods.3. The apparatus according to claim 1 further comprising a third bath,wherein a bath medium of the third bath is configured to be maintainableat a medium temperature T_(MEDIUM), wherein the transfer is configuredfor keeping the reactor to be in the third bath to attain apredetermined medium target temperature T_(MT), and the T_(MEDIUM) isequal to the T_(MT) or offset from the T_(MT).
 4. The apparatusaccording to claim 1 further comprising a fourth bath, wherein a bathmedium of the fourth bath is configured to be maintainable at atemperature T_(AP) to perform an additional process for the reactorbefore the thermal cycling, the additional process being one selectedfrom the group consisting of a) reverse transcription-polymerase chainreaction (RT-PCR), b) hot start process and c) isothermal amplificationreaction, wherein, the transfer means is configured for keeping thereactor to be in the fourth bath to attain an additional process targettemperature T_(APT), the T_(AP) is equal to the T_(APT) or offset fromthe T_(AP).
 5. The apparatus according to claim 1, wherein the bathmedium in the first bath is maintainable above 100 degrees Celsius. 6.The apparatus according to claim 1, wherein the bath medium in thesecond bath is maintainable below room temperature.
 7. The apparatusaccording to claim 1, where the transfer means is calibrated to initiatelift-off of the reactor from the two baths when the reactor reaches afirst lift-off temperature that is lower than the T_(HT) and a secondlift-off temperature that is higher than the T_(LT), in order tocompensate for operational electro-mechanical delays unwantedly causingover heating or over cooling of the reactor.
 8. (canceled)
 9. (canceled)10. (canceled)
 11. (canceled)
 12. (canceled)
 13. (canceled)
 14. Theapparatus according to claim 1, wherein, a first offset between theT_(HIGH) and the T_(HT) is within a range of 1-400 degree Celsius and asecond offset between the T_(LOW) and the T_(LT) is within a range of1-100 degree Celsius.
 15. The apparatus according to claim 1, wherein,the bath medium in the first bath is a first liquid added to a secondliquid, a boiling point of the second liquid being higher than a boilingpoint of the first liquid.
 16. The apparatus according to claim 15,wherein, a boiling point of a mixture of the first liquid and the secondliquid is higher than 100 degree Celsius.
 17. The apparatus according toclaim 1 further comprising bath covers on the two baths, an opening onthe covers for keeping the reactor to be in the two baths and closingafter the reactor is removed from the two baths.
 18. (canceled) 19.(canceled)
 20. (canceled)
 21. A method for thermal processing nucleicacid in a thermal profile, comprising: employing a reactor holder forholding a reactor, wherein, the holding reactor accommodates, reactionmaterial containing the nucleic acid; employing an apparatus comprisingtwo baths including a first bath and a second bath; maintaining bathmediums in the first bath at temperature T_(HIGH) and in the second bathat temperature T_(LOW); and employing a transfer means in the apparatusto keep the reactor to be in the two baths in a plurality of thermalcycles to alternately attain: a predetermined high target temperatureT_(HT), and a predetermined low target temperature T_(LT); and adaptingto a temperature-offset feature defined by at least one conditionselected from the group consisting of: a) the T_(HT) is lower than theT_(HIGH), and b) the T_(LT) is higher than the T_(LOW) the transfermeans being operable by at least one mode selected from the groupconsisting of: a temperature guided motion controlling means (TeGMCM)that is operable based on a real-time temperature as sensed by a reactortemperature sensor during thermal cycling, and a time guided motioncontrolling means (TiGMCM) that is operable based on time-periods forwhich the reactor are allowed to be in the two baths; the bath mediumsin the two baths are in gaseous form, liquid form, solid form, powderform or combination thereof.
 22. (canceled)
 23. (canceled)
 24. Themethod according to claim 21 further comprising maintaining the bathmedium in the first bath above 100 degrees Celsius.
 25. The methodaccording to claim 21 further comprising maintaining the bath medium inthe second bath below room temperature.
 26. The method according toclaim 21 further comprising calibrating the transfer means to initiatelift-off of the reactor from the two baths when the reactor reaches afirst lift-off temperature that is lower than the T_(HT) and a secondlift-off temperature that is higher than the T_(LT) to compensate foroperational electro-mechanical delays unwantedly causing over heating orover cooling of the reactor.
 27. (canceled)
 28. (canceled) 29.(canceled)
 30. (canceled)
 31. (canceled)
 32. (canceled)
 33. (canceled)34. The method according to claim 21 further comprising adding a secondliquid to a first liquid in the bath medium in the first bath, a boilingpoint of the second liquid being higher than a boiling point of thefirst liquid.
 35. The method according to claim 34, wherein, a boilingpoint of a mixture of the first liquid and the second liquid is higherthan 100° C.
 36. (canceled)
 37. (canceled)
 38. (canceled)
 39. (canceled)